Transitional-Tapping AFM: Probing Stochastic Molecular Dynamics at Sub-kBT Precision

Transitional-Tapping AFM: Probing Stochastic Molecular Dynamics at Sub-kBT Precision | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Transitional-Tapping AFM: Probing Stochastic Molecular Dynamics at Sub-kBT Precision Seonghwan Kim, Arindam Phani, Allegra Behr, Balasubramanian Srinivasan, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6185843/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Conventional tapping-mode atomic force microscopy (AFM) is limited in its ability to resolve time-varying interaction forces during rapid and stochastic molecular transitions at a sample pixel while scanning. Resolving these transitions is essential for tracking molecular assembly and rearrangements in real-time. We introduce Transitional-tapping ( Tt ) AFM, a technique that achieves this by directly measuring sample stochasticity from higher harmonic tip interaction forces. Tt-AFM harmonizes tip-sample motion by fine-tuning tip oscillations to sustain continuous engagement during scanning. We demonstrate its capability by uncovering transient native-like protein intermediates, that align remarkably with AlphaFold and Protein Data Bank predictions. The imaged intermediates retain native folds but undergo subtle chain rearrangements, persisting as ‘rest-stops’ for nanoseconds along the folding landscape to stabilize. Tip dynamics reveal that shear-thinning guides folding through sub-diffusive Lévy-jump kinetics. Transitional-tapping fundamentally transforms AFM’s capability to probe stochasticity and intermediate energetics at sub-k B T precision, unlocking new frontiers in soft-matter AFM. Physical sciences/Nanoscience and technology/Techniques and instrumentation/Imaging techniques Biological sciences/Biotechnology/Nanobiotechnology/Applications of AFM Atomic Force Microscopy (AFM) Transitional-tapping Protein folding Metastable protein intermediates Molecular self-assembly Conformational dynamics Sub-diffusive Lévy kinetics Folding energy landscapes Full Text Additional Declarations Yes there is potential Competing Interest. The Transitional TappingTM method is Patent pending (US Patent App. 18/282,136). Supplementary Files SupplementaryMaterialManuscriptPhanietalMar3.docx supplementary information Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6185843","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":429708856,"identity":"600f37f2-adb4-40af-b151-e18739abe4d8","order_by":0,"name":"Seonghwan 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